estrogen and progesterone receptor proteins in breast cancer

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Bioehimiea et Biophysica Acta, 560 (1979) 457--486 © Elsevier/North-Holland Biomedical Press

BBA 87062

E S T R O G E N AND P R O G E S T E R O N E R E C E P T O R PROTEINS IN BREAST CA N CE R

DEAN P. EDWARDS, GARY C. CHAMNESS and WILLIAM L. McGUIRE *

Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78284 {U.S.A.)

(Received December 13th, 1978)

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457

II. Cytoplasmic estrogen receptor in human breast cancer . . . . . . . . . . . . . . . . . . . . 459 A. Assay methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 B. Clinical results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

III. Nuclear estrogen receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

IV. Progesterone receptor in human breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . 464 A. Estrogen regulation of progesterone receptor in breast cancer cells . . . . . . . . . . . 464 B. Progesterone receptor assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 C. Clinical results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466

V. Mechanisms of estrogen action in a human breast cancer cell line (MCF-7) . . . . . . . . . 468

VI. Antiestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 A. Experimental animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 B. Human breast cancer cells in tissue culture . . . . . . . . . . . . . . . . . . . . . . . . . 475 C. Clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

VII. Current concepts and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

I. Introduction

It has been k n o w n for some t ime that cer tain h u m a n breast cancers regress af ter endo-

crine manipula t ions . These manipula t ions include ovar iec tomy, ad rena lec tomy , hypo-

p h y s e c t o m y , t r e a t m e n t wi th an t ies t rogens , or admin is t ra t ion o f pharmacologica l doses o f

es t rogens or androgens . In general , about 30% o f breast cancer pa t ien ts will exper ience an

object ive remission o f their disease given one o f these t r ea tmen t s , and it seems likely,

* To whom reprint requests should be addressed. Abbreviation: DMBA, 7,12-dimethylbenz(a)anthracene.

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though not totally proven, that most of these same patients would respond to any of these treatments [1]. Until recently, however, there has been no reliable way to determine which cancers are hormone dependent without an actual treatment trial. Our goal here is to review the contribution of steroid hormone receptor studies to the resolution of this problem, and to discuss our current knowledge of steroid hormone action in the control of breast cancer cells.

The first steroid receptor to be demonstrated was the estrogen receptor by Toft and Gorski in 1966 [2]. Since then, estrogen and progesterone receptors have been described in appropriate target tissues [3-5] , including normal mammary gland [6-9] and some breast tumors [10-12]. Steroid receptor proteins are found principally in the cytoplasm of target tissues for their respective hormones. They display ligand specificity by binding with very high affinity only those hormones of their own class which exhibit biologic activity. Upon binding with hormones, receptors are translocated to the cell nuclei where they initiate certain biochemical activities which ultimately lead to the physiological expressions characteristic of the particular hormone. Much work has been done on the early events which follow steroid hormone stimulation, utilizing most widely, the immature rat uterus as a model system for studying estrogen mediated growth and development of target tissues. A temporal sequence of events occur in estrogen mediated growth, which includes stimulation of macromolecular synthesis (RNA, protein and DNA) and various metabolic activities. For a detailed discussion of the biosynthetic and physiological events initiated by estrogens in target tissues, see the review by Katzenellenbogen and Gorski [ 13]. Although little is definitely known of the immediate mechanism of receptor action in the nucleus, it has been hypothesized that these actions follow from the binding of the receptor-steroid complex to specific chromatin 'acceptor' sites [14,15]. In at least some cases, the receptor-hormone complex then disappears from the nucleus without returning to the cytoplasm; replenishment of receptors in the cytoplasm requires new receptor synthesis [16]. As will be discussed later, this nuclear depletion or 'processing' of the receptor-steroid complex may be a necessary step in receptor action. Steroid receptor mechanisms have been extensively discussed elsewhere and the reader should refer to these excellent reviews for more detailed information [3-5] .

It was Jensen in 1967 [17] who first proposed that the restriction of receptors to hormone target cells might extend to breast cancers, thus suggesting a way to distinguish hormone dependent from autonomous tumors without a direct trial of endocrine therapy. The idea was investigated in several laboratories in several countries, and an international workshop which considered the data in 1974 was able to conclude that very few tumors without detectable cytoplasmic estrogen receptor responded to any endocrine manipulation, while nearly 60% of those with measurable estrogen receptor did have a favorable response [12]. Clearly, however, this approach leaves a considerable gap in our ability to predict hormone dependence of a particular tumor. In order to fill this gap, many studies of a more detailed nature on steroid hormone action and of other steroid receptors in breast tumors have been undertaken.

There are several available model systems for studying hormone action in mammary tumors, and two of these have been particularly productive. The first is the dimethyl- benzanthracene (DMBA)-induced mammary carcinoma in Sprague-Dawley rats, originally described by Huggins et al. [18]. The majority of these tumors are hormone dependent, but since each tumor represents a separate malignant transformation, many different types arise. This is clearly an advantage for certain types of study. In spite of the considerable progress which has been made using rat DMBA-induced tumors, however, the sys-

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tern has two distinct disadvantages besides nonuniformity of tissue. First, DMBA induced tumors fail to metastasize. Secondly, extensive research has led to the conclusion that the chief regulation of DMBA tumor growth in rats is provided by prolactin, with estrogen playing a necessary but secondary role [19], while in human breast cancer most studies indicate that prolactin is much less important, leaving estrogen control predominant [20]. A model system of human origin might therefore yield more relevant information on hormone dependence in human tumors.

Such a system is available since the recent establishment of hormone responsive human breast cancer cell lines in tissue culture. The MCF-7 cell line, derived from the pleural effusion of a patient with metastatic breast cancer [21 ], seems particularly well suited for studies on hormone control of human breast cancer since the cells contain receptors for all four principal classes of steroid hormones [22,23]. As a model for study, cells grown in continuous tissue culture also provide the distinct advantage of permitting experiments in a hormonally controlled environment and with a homogeneous population of cells, which is possible in no other kind of system. The body of knowledge derived from studies with MCF-7 cells is growing rapidly. Much of this review will therefore relate to work from our laboratory on estrogen receptor mechanisms and actions of antiestrogens, as well as the significance of progesterone receptors, using primarily MCF-7 cells. Results from rat DMBA-induced tumors will be included where necessary. Much consideration will also be devoted, however, to current clinical results, since our ultimate goal is refine- ment of the ability to control tumor growth by endocrine manipulations and to develop accurate predictive tests in advance of endocrine therapies.

II. Cytoplasmic estrogen receptor in breast cancer

IIA. Assay methods There are several procedures available for measurement of cytoplasmic estrogen recep-

tors in mammary tissues [11,24-27]. The receptor can be quantitated by demonstration of specific 8 S and 4 S binding of [3 H]estradiol on sucrose density gradients or by a standard dextran coated charcoal assay. We have compared assays in 29 human tumor specimens and have concluded that quantitatively, both assays are equivalent [26]. The sucrose density gradient assay has the advantage, however, of demonstrating an 8 S peak, which is a qualitative characteristic of cytoplasmic estrogen receptors.

Assays based on protamine sulfate precipitation of the receptor have been developed to measure both unbound and hormone-bound receptor from cytoplasmic [28] and nuclear [29] extracts. The receptor is precipitated with a protamine solution and the solid phase protamine-receptor complex is then incubated with radioactive estradiol. Incuba- tion at 30 or 37°C permits exchange of any previously bound endogenous ligand, while at 4°C only unoccupied receptor becomes labeled. The combination of these assays has the unique advantage of using only one technique to assess both free and bound estrogen receptor sites in cytosol and nuclear fractions. It was thought that this procedure might prove useful for premenopausal cancer patients who might have high levels of plasma estrogens that would transfer cytoplasmic estrogen receptors to nuclear sites, making then inaccessible to assay by sucrose density gradients or dextran coated charcoal. Since the presence of free cytoplasmic estrogen receptors in tumors has prognostic value in helping to predict the proper type of treatment for breast cancer patients, those premenopausal women who have estrogen receptors masked by endogenous estrogens might not be given treatment that would be of greatest benefit.

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The protamine sulfate assay for measuring bound and free estrogen receptor sites has been used successfully with human breast cancer cells in tissue culture [30]. We have found, however, that proteolytic activities in extracts from nuclear pellets of actual human breast tumor biopsies interferes with measurement of nuclear estrogen receptor by the protamine assay. The problem of nuclear protease activity can be avoided by using an hydroxylapatite assay, in which the receptor is first adsorbed to hydroxylapatite and then inducated with hormone [31]. The proteolytic activity is apparently not adsorbed by the hydroxyapatite, and the assay of nuclear estrogen receptors is therefore unaffected. The hydroxylapatite assay has also proven useful for measuring total receptors, hormone- bound and free, in both DMBA rat mammary tumors and human tumor specimens by exchange of endogenous ligand at elevated temperatures [32].

We have recently introduced a micro-assay for measuring estrogen receptors in human tunror specimens [33]. The micro-assay is based on hydroxylapatite pre-precipitation of the receptor and is a reliable method for the assay of estrogen receptors in very small tumor samples, requiring only 30% of the tissue material used for the standard dextran coated charcoal assay.

liB. Clinical results

Jensen's orginal suggestion [17] that the presence of estrogen receptors in a human breast tumor biopsy might indicate whether the tumor was hormone responsive and thus able to regress with appropriate endocrine therapy has been evaluated by a number of laboratories [ 12].

Estrogen receptor data from our own laboratory is presented in Table 1. Patient breast tumor biopsies were analyzed for the presence or absence of cytoplasmic estrogen receptors by a dextran-coated charcoal assay [26]. Objective tumor regression was evaluated in each patient following various types of endocrine therapies, both ablative and additive. Patients with estrogen receptor negative tumors rarely responded to endocrine treatments, whereas 57% of the patients classified as having estrogen receptor positive tumors responded to endocrine therapy.

Classifying patient's tumors as estrogen receptor-positive and estrogen receptor-neative is arbitrary and in most cases probably reflects the lower limit of sensitivity of a particular receptor assay. This approach for classification of patients has drawn criticism, especially in view of reports that all breast tumors may contain some estrogen receptors [34, 35] and that important differences among patients may be quantitative rather than qualitative with regard to estrogen receptors. We have therefore examined whether there might be a correlation between objective tumor response to endocrine therapy and the concentration of estrogen receptor sties. The data shown in Table II indicates that intermediate

TABLE I

ESTROGEN RECEPTOR IN METASTATIC BIOPSIES AND OBJECTIVE RESPONSE TO ENDO- CRINE THERAPY

Estrogen receptor Estrogen receptor negative positive

Ablative 1 / 19 25/43 Additive 1/14 19/34

Total 2/33 = 6% 44/77 = 57%

TABLE II

ESTROGEN RECEPTOR VALUES AND OBJECTIVE RESPONSE TO ENDOCRINE THERAPY

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Estrogen receptor Response (fmol/mg protein)

<3 2/33 = 6% 3 100 24/52 = 46%

101-1000 21/26 = 80%

estrogen receptor values are associated with an intermediate response rate, while patients with higher estrogen receptor values respond with a much higher rate. This study indicates that quantitative interpretation of estrogen receptor data is of greater predictive value for the success of endocrine therapy than qualitative assessment alone. Heuson et al. [36] also examined the correlation between estrogen receptor concentration and patient tumor response to endocrine therapy. They performed a regression analysis on information obtained from 25 patients and found that among 12 variables known to be of prognostic value, estrogen receptor concentration was the most significant.

The possible explanations for the imperfect correlation between cytoplasmic estrogen receptor data and breast tumor response to endocrine therapy are numerous. First, breast tumors may contain a continuum of estrogen receptor concentrations, ranging from trace amounts to very high concentrations. Data noted above suggests that it may be more appropriate to select endocrine therapy based on a quantitative rather than qualitative assessment of estrogen receptors. Second, clinical results with regard to the biological behavior of the tumor in response tQ endocrine manipulation may be misleading because of the rigid criteria for objective tumor regression. For example, endocrine therapy may not kill all the cells in a tumor but may simply inhibit cell replication such that the balance between replication and cell death and shedding will be shifted, resulting in a smaller but stable tumor. Such a tumor clearly responds to hormonal manipulation, but by strict clinical definition that manipulation may fail to kill enough cells to be considered an effective treatment. Third, if a patient were producing high levels of estrogens, we would anticipate appreciable translocation of cytoplasmic estrogen receptors into the cell nucleus. Thus routine utilization of exchange assays for detecting estrogens endogenously bound to nuclear receptors may improve the predictive value of estrogen receptor data. Finally, although a tumor may contain appreciable levels of cytoplasmic estrogen receptors, there may be a defect in the biochemical machinery of estrogen action, distal to receptor-ligand binding, rendering the tumor insensitive to estrogen. In such cases, a measurable end product of estrogen action, as an additional biochemical marker, would improve estrogen receptor data as a prognostic indicator of tumor response to endocrine therapy.

Regardless of the reasons for the imperfect correlations between cytoplasmic estrogen receptors data and tumor response to endocrine therapy, the response rate in those patient tumors containing cytoplasmic estrogen receptors is substantial, so that measuring cytoplasmic estrogen receptors is undoubtedly of considerable value in predicting the behavior of particular tumors to endocrine manipulations. Other kinds of biochemical data are needed, however, if we are to be able to identify a priori those tumors containing cytoplasmic estrogen receptors which will fail to respond to hormonal therapies.

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lIl. Nuclear estrogen receptor

A contributing factor to the variable patient tumor response to endocrine therapies may be the inherent limitations of conventional estrogen receptor assays, which detect only unfilled cytoplasmic receptor sites. Since circulating estrogens entering the target cell serve to bind and translocate receptors to the nucleus, measuring only cytoplasmic receptors creates the possibility for underestimating estrogen receptor levels and even for reporting false negative results. Preliminary evidence that endogenous estrogen may influence cytoplasmic receptor levels is provided by the observation that postmenopausal patients tend to have higher concentrations of cytoplasmic estrogen receptors in their tumors than premenopausal patients [12,37-39] and from the fact that Maass et al. [40] have found lower levels of cytoplasmic receptor when plasma estrogen concentrations are high. Additionally, Sakai and Saez [41], using an estradiol exchange assay, to measure both filled and unfilled receptor sites in the cytoplasm and total cellular extracts of human breast tumors, have found a positive correlation between the degree of receptor occupancy and the concentrations of circulating estrogens in premenopausal patients. When the number of estrogen receptor sites occupied by endogenous hormone was determined and expressed as the percentage of total sites (filled and unfilled), the percentage was found to range between 0 and 36%, indicating that even at high levels of circulating estrogens, receptor sites in human tumors are never fully saturated [41]. Fishman et al. [42] also showed that the failure to detect estrogen receptors in some tumors by the conventional cytosol assay, is not due to complete saturation of binding sites by high endogenous estrogen concentrations, since studies in 129 human breast tumor samples revealed that high tissue concentrations of endogenous estrogens were not related to low or negative cytoplasmic receptor levels. Since receptors are continually replenished during estrogen stimulation, it may not be surprising to find unfilled cytoplasmic estrogen receptor sites even in the presence of high levels of endogenous estrogen.

To pursue these questions more directly and to consider whether some tumors contain receptors which are incapable of translocating to the nucleus, rendering the tumor cells unresponsive to estrogens, we have now utilized the hydroxylapatite estradiol exchange assay, to simultaneously measure both unfilled cytoplasmic and total nuclear estrogen receptor sites (filled and unfilled) in a series of human breast tumors [32]. Table III shows the cytosol and nuclear estrogen receptor values obtained in 28 human tumor biopsies. Some tumors were found to contain both estrogen-bound and free nuclear receptors. The percentage of tumors in this study containing nuclear estrogen receptors was roughly equivalent to the percentage containing cytoplasmic receptors. Furthermore, in every tumor containing nuclear receptor, this was accompanied by detectable cytoplasmic receptor though no quantitative relationship between nuclear and cytoplasmic receptor content was apparent. Three tumors in this series contained only free cytoplasmic receptor sites, providing at least a preliminary suggestion that receptor translocation may indeed be defective in some tumors. Studies with a much larger number of tumors are needed to substantiate this. Thus, in agreement with the findings of other investigators [41,42], our studies indicate that estrogen receptors in human breast tumors may never be fully saturated, even in the presence of plasma estrogens sufficient to cause translocation of receptors to the nucleus. From this preliminary study it has not been possible, therefore, to explain the dependency of the 6% of tumors which lack detectable cytoplasmic estrogen receptors yet respond to endocrine therapy, by the presence of only hormone-bound nuclear receptors. Our results also indicate the unlikelihood of reporting

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TABLE III

CYTOSOL AND NUCLEAR ESTROGEN RECEPTOR IN HUMAN BREAST CANCER

Tumor fmol/mg DNA

Cytosol * Nuclear extract

Occupied sites Free sites

1 20 670 286 206 2 5 469 195 208 3 4 156 513 70 4 3 526 284 177 5 2 761 411 870 6 2 160 87 14 7 1 781 364 158 8 1 483 450 141 9 1 481 243 174

10 1 151 309 17 11 1 030 533 0 12 899 0 225 13 847 501 242 14 547 0 0 15 294 165 256 16 233 0 0 17 45 0 0 18 36 29 29 19 25 0 35 20 22 81 88 21 14 9 2 22 0 0 0 23 0 0 0 24 0 0 0 25 0 0 0 26 0 0 0 27 0 0 0 28 0 0 0

* Cytosol estrogen receptor and unoccupied sites were measured by a single-dose hydroxyapatite assay at 4°C for 6 h. Occupied nuclear sites were measured by the difference between total sites (30°C for 5 h ) and free sites. (From Garola and McGuire [32]).

totally false estrogen receptor-negative data from the results of conventional cytosol

receptor assays. The fact that a portion of the total estrogen receptor in the nucleus of some tumors

was unoccupied by estrogen, led us to speculate that free nuclear receptor may be able to

stimulate tumor growth in the absence of estrogen [30]. If this were true, tumors con-

taining appreciable amounts of free nuclear receptor might not respond to endocrine

ablative procedures designed to reduce endogenous estrogens. The observation that some

breast tumors contain unfftlled nuclear receptors for estrogen has been extended by Panko

and MacLeod [43] in a study taking care to ensure that nuclear preparations were free of

cytoplasmic contamination and that previously bound endogenous estrogen was not

stripped from the receptor during extraction in the high-ionic strength buffers. Of 139

tumor specimens, they found the incidence o f breast cancers contai13ing amounts of free

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nuclear estrogen receptor greater than 7 fmol/mg protein was almost 30%. This is about the same percentage of estrogen receptor-positive tumors that do not respond to ablative therapy. Laing et al. [44] have reported that the incidence of estrogen bound nuclear receptors in human breast cancers is 39%, a value roughly equivalent to the incidence for free nuclear receptors reported by Panko and MacLeod [43]. Laing et al. [44] however, measured 'estrogen-bound nuclear receptor' by incubating for 18 h at 4°C. From our own experience we would suggest that bound estradiol does not freely exchange at 4°C and that they have, in fact, measured unfilled receptor sites.

It is evident from these preliminary studies that conventional cytoplasmic estrogen receptor assays may underestimate total estrogen receptor sites in patient tumors. Since it appears that a quantitative assessment of estrogen receptors may be of greater predictive value than the mere presence or absence of receptors, additional routine measurements of nuclear estrogen receptors may be of potential value. The finding that some tumors contain unbound nuclear receptor for estrogen may also warrant the use of nuclear estrogen receptor assays in the management of breast cancer, since if free nuclear receptors stimulate tumor growth in the absence of estrogen, then those breast cancers containing free nuclear receptors might represent a group in which antiestrogen treatment would be the more appropriate form of endocrine therapy.

IV. Progesterone receptor in human breast cancer

IVA. Estrogen regulation of progesterone receptor in breast cancer cells The fact that not all mammary carcinomas containing estrogen receptor respond to

endocrine therapy has led to the idea that in such tumors, defects in estrogen stimulated pathways may exist, distal to the initial receptor binding and translocation steps. In such cases, a product of estrogen action would be a more useful biochemical marker of the tumor's hormone responsiveness. Since progesterone receptor in the uterus is known to be controlled by estrogen [45-47] , we have hypothesized that the presence of progesterone receptor in breast tumors containing estrogen receptor would indicate that the entire sequence of events involving estrogen action was functional and that the tumor was capable of synthesizing at least one end product of estrogen action. The hypothesis assumes, however, that progesterone receptor levels in breast tumors, like those in the uterus, are regulated by estrogen, and that the estrogen stimulated pathways regulating tumor growth and progesterone receptor synthesis are not independent.

That the first condition is true was shown first by our studies with the DMBA-induced rat mammary tumor, in which estrogen was demonstrated to exert acute control over the modulation of progesterone receptor levels [48]. The same observation has been con- firmed by other investigators [49]. These experiments were followed by examination of the effects of estrogen on progesterone receptor synthesis in the MCF-7 cell line [16]. Cells grown on estrogen-free medium contained a low but consistent basal level of progesterone receptor. Progesterone receptor levels in cells grown 4 days on medium containing estradiol increased 3 to 4-fold above basal levels. Fig. 1 shows the sucrose density gradient profile of progesterone receptor stimulation by estradiol in MCF-7 cells. In other experiments, estradiol was observed to stimulate progesterone receptor in a dose-dependent manner, maximal stimulation occurring at 0.1 nM, which is a nearly physiological level for circulating estrogen in women. This is the first demonstration that progesterone receptors in human breast cancer cells are regulated by the action of estrogens. This study has also been important in showing that the MCF-7 breast cancer cells, which contain an

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estrogen receptor, are capable of synthesizing a specific end product of estrogen action. Furthermore, demonstrating the control of progesterone receptor levels by estrogen in human breast cancer cells provides credibility to the working hypothesis that the simultaneous presence of progesterone receptor and estrogen receptors in a breast tumor biopsy indicates the presence of a complete pathway capable of responding to circulating estrogens.

IVB. Progesterone receptor assays Two major problems in the detection of specific progesterone receptor proteins have

been that progesterone binds to certain plasma proteins such as corticosteroid-binding globulin (CBG) with high afffmity, and that it dissociates rapidly from its specific progesterone receptor binding sites. Thus, due to interference with plasma proteins which may contaminate a specimen and to the lability of the receptor-hormone complex, demonstration of progesterone receptor using native progesterone as ligand has proven to be difficult. Terenius [24] however, by a dextran coated charcoal method and using excess unlabeled hydrocortisone to compete out binding to CBG was able to demonstrate Jail]pro. gesterone binding to progesterone receptor in human and rat mammary carcinomas. Pichon and Milgrom [50] have also successfully employed [3H]progesterone to measure progesterone receptor in human breast tumors by including 30% glycerol into incubation buffers and sucrose gradients and by incubating with excess hydrocortisone. Glycerol apparently stabilizes the receptor-progesterone complex, thus decreasing it's dissociation rate constant. To circumvent these problems a synthetic radiolabeled ligand, R5020, was developed which is reported to bind to progesterone receptors in the rat uterus with high

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affinity, not to bind to CBG, and to exhibit a slow rate of dissociation from progesterone receptor thus stabilizing the receptor-ligand complex [51]. With the synthesis of 3H- labeled ligand R5020, progesterone receptors have now been unequivocally demonstrated in normal lactating mammary glands [9,52], certain experimental rat mammary tumors [48,53] and human breast cancer [22,54,55].

[3H]Progesterone and ligand [3H]R5020 have been used in our laboratory to demonstrate progesterone receptor in human breast tumor specimens [56]. Ample evidence has been provided to demonstrate that [3H]progesterone and ligand [3H] R5020 bind to iden- tical receptor sites, probably the best being that ligand R5020 binding is sensitive to estrogen action in parallel with estrogen stimulation of progesterone-binding sites [56]. We have compared sucrose gradient profiles of specific [3H] progesterone and ligand [3H]- R5020 binding to the same human tumor cytosol. Progesterone bound protein sedimented as a single 4 S entity, while bound ligand R5020 sedimented in both 8 S and 4 S regions of the gradient. Competition studies revealed that 91% of the 4 S [3H]progesterone binding was suppressed with excess unlabeled progesterone, yet competition with ligand R5020 showed that only 30% of this binding was to progesterone receptor. Excess unlabeled ligand R5020 and progesterone, however, completely inhibited binding of ligand [3H]R5020 in the 8 S peak. Thus, ligand R5020 binding in the 8 S regions of sucrose density gradients undoubtedly represents specific progesterone receptor while it is questionable from these studies whether specific 4 S binding represents progesterone receptor.

We have further analyzed the true nature of specific ligand R5020 binding detected by sucrose density gradient centrifugation by comparison of this with several different assay methods for progesterone receptor. Gradient profiles of ligand R5020 binding obtained by the standard 16 h centrifugation in a swinging bucket rotor were compared to those observed by 2 h centrifugation in a vertical tube rotor. Use of the swinging bucket rotor by comparison to the vertical tube rotor caused an under estimation of 8 S binding, presumably due to partial dissociation of the binding complex during the prolonged run time. The specific 4 S component was shown probably not to be progesterone receptor since the binding complex in the 8 S gradient fractions was absorbed to hydroxyapatite and the binding component in the 4 S fractions of most tumor samples failed to do so. To confirm further that the 4 S binding component is not progesterone receptor, values for progesterone receptor sites derived from Scatchard analysis of dextran coated charcoal and hydroxyapatite assays correlate very well with the number of binding sites in the 8 S component of the gradient but not with the total sites in the binding profile (8 S + 4 S). We suggest therefore, that progesterone receptors in human tumors be assayed by sucrose density gradient centrifugation in a vertical tube rotor and that the specific 4 S binding component should not be considered to be progesterone receptor (Powell and McGuire, unpublished data).

IVC. Clinical results Simultaneous measurements of estrogen receptors and progesterone receptor in a large

number of human mammary tumors have been performed in several laboratories. Our own series [56] shows that tumors containing no estrogen receptors are not likely to contain any progesterone receptor (9%). A tumor with measurable estrogen receptors, however, is very likely to also contain progesterone receptor (74%)..This strong correlation suggests, as we have previously shown in experimental rat mammary tumors and in human breast cancer cells in culture, that in patient tumors progesterone receptor is regu-

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TABLE IV

COMPARISON OF ESTROGEN RECEPTOR CONTENT AND THE NUMBER OF PROGESTERONE RECEPTOR POSITIVE BREAST TUMORS

Estrogen receptor Progesterone receptor No. of patients (fmoles/mg protein) % positive

<3 5 27 3 10 25 8

1 1 - 100 63 29 100-1000 82 22

lated by estrogen acting through its receptor. We have also found that the likelihood of a

tumor containing measurable progesterone receptor increases as the estrogen receptor content in the tumor increases (Table IV).

Table V is a compilation of all the presently available data correlating estrogen receptor and progesterone receptor distribution in human breast tumors with clinical response to endocrine therapy. It includes published data [56-59] as well as our own current data and some obtained by personal communication. Since the patient selection, receptor assay methods, and criteria for objective tumor response are probably different for each

investigator, it is not appropriate to pool the data to arrive at definitive conclusions. How- ever, because of the potential value of progesterone receptor as a marker for tumor sensitivity to estrogens, we feel it is important to consider these preliminary results from a number of different laboratories. It is clear that patients with tumors containing both progesterone receptor and estrogen receptors do have the most favorable response rates. From these preliminary studies, measurements of progesterone receptor in patient tumors

definitely improves data from estrogen receptor assays alone as a prognostic indicator of a tumor's sensitivity to estrogens. A sizeable group of patients, however, have been identified as estrogen receptor-positive/progesterone receptor-negative, yet respond to endocrine therapies. We would have predicted that these patients would have defective machinery for estrogen action and thus would not respond.

TABLE V

OBJECTIVE RESPONSE TO ENDOCRINE THERAPY (231 CASES) BY RECEPTOR CONTENT

ER, estrogen receptor; PgR, progesterone receptor.

Reference ER-, PgR- ER-, PgR+ ER+, PgR- ER+, PgR+

McGuire et al. [56] 0/11 -- 7/17 13/16 Degenshein et al * 0/ 7 1/1 1/ 9 16/18 King et al. ** 1/ 5 1/2 1/ 7 8/ 9 Skinner etal.*** 5/17 -- 0/ 3 4/ 7 LeClercq et al. [571 - 0/1 1/ 3 1/ 3 Matsumoto et al. [58] 2/20 0/1 9/25 12/20 Young et al. [59] 1/ 3 1/1 I/ 7 13/18

Totals 9/63 = 14% 3/6 20/71 = 28% 67/91 = 74%

* Personal communication. * * King, R.J.B., Redgrave, S., Rubens, R.D., Millis, R. and Hayward, J.A., manuscript in preparation.

*** Skinner, L.G., Barnes, D.M. and Ribeiro, G.G., manuscript in preparation.

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There are several possible explanations for this apparent contradiction. The most ob- vious is that false negatives were reported for progesterone receptor in these patients. For instance, dissociation of the ligand from the 8 S receptor during the standard 16 h centrifugation may have resulted in underestimation of progesterone receptor levels. False negative progesterone receptor could be reported, however, for other reasons. For one, it is possible that at the time of biopsy, there was not sufficient endogenous estrogen to stimulate progesterone receptor synthesis. Thus it may be useful to prime a patient with estrogen prior to biopsy. While theoretically this may be possible, in practice this may be regarded as a dangerous procedure in a premenopausal patient for fear of aggravating an estrogen-sensitive tumor. Also, it must be considered that if the biopsy were taken during the luteal phase of the cycle, there may have been sufficient endogenous progesterone to fill cytoplasmic progesterone receptor and/or translocate it to the nucleus. In such cases, measuring only unfilled cytoplasmic progesterone receptor as we routinely do would give false negative information. There is now evidence to show that the incidence of detectable progesterone receptor decreases dramatically in patients with elevated blood progesterone levels [60]. Thus, the proper tinting of patients for progesterone receptor assays may be more critical than selection for estrogen receptor assays.

As indicated in Table V, there are also some patients identified as estrogen receptor- positive/progesterone receptor-positive which nevertheless fail to respond to endocrine therapies. We must therefore consider that our hypothesis may not be fully correct. The pathways for estrogen regulation of tumor growth and progesterone receptor synthesis may be independent. Alternatively, estradiol may not be the predominant hormone regulating growth in every hormone-dependent breast tumor, such that groups of patients may contain tumors in which growth is regulated by other hormones. With the current work on progesterone receptor in mammary carcinomas, it should be possible in the near future to more accurately correlate the presence of progesterone receptor with a particular tumor's growth responsiveness to estrogens.

V. Mechanisms of estrogen action in a human breast cancer cell line (MCF-7)

Because clinical studies and animal studies have not completely defined the hormonal mechanisms regulating growth and development of hormone-responsive mammary tumors, we have turned to human breast cancer cells in permanent tissue culture for further studies. The most extensively studied of these cells is the MCF-7 line, which unlike nine other lines tested, was found to contain all four receptor classes studied (estrogen, glucocorticoids, progestins and androgens) [22,23]. Thus, it is an excellent model for studying hormone mechanisms of tumor growth control as well as the interrelationships between hormones.

MCF-7 cells are biologically responsive to estrogens. Growth studies have shown that addition of physiological concentrations of estradiol to cells maintained in estrogen-free medium results in a significant increase in the number of cells above controls by the eighth day of culture [61]. These cells are not, however, fully dependent on estrogens for growth, since cells are well maintained and continue to grow in the absence of estrogens. Nevertheless, Lippman's group [62-64] has demonstrated that macromolecular synthesis in the MCF-7 cell line is extremely sensitive to estrogen. Concentrations as low as 0.02 nM estradiol stimulated protein, RNA, and DNA synthesis, and the relative binding affinity of estrogens for their receptor was found to be proportional to their potency in stimulating thynridine incorporation. Thymidine kinase, a potential rate limiting enzyme of the

469

salvage pathway for deoxynucleotide biosynthesis, was also shown to be regulated as a function of estrogen concentration in MCF-7 cells [65]. Dose curves of estrogen binding to receptor and stimulation of thymidine kinase activity paralleled each other, suggesting that estrogen stimulation of MCF-7 functions is mediated by estrogen interaction with its receptor.

Recent studies from our own laboratories demonstrate that lactate dehydrogenase, which is believed to be related to the degree of malignancy of a tumor [66] and has the potential to influence metabolic functions that may be crucial to growth of the cell, is also influenced by estrogens. We have found that physiological concentrations of estradiol stimulate lactate dehydrogenase in MCF-7 cells two-fold above controls in a dose dependent manner [67]. Thus lactate dehydrogenase may be a useful marker for studying estrogen action in breast cancer cells.

We have also shown that estrogen receptor can regulate the levels of DNA polymerase a activity in MCF-7 cells (Edwards and McGuire, unpublished data). Cells were grown initially for 5 days on the antiestrogen nafoxidine, to arrest growth and the medium was then changed to either control medium, medium plus nafoxidine or medium plus 0.01/2M estradiol. Estradiol stimulated cytosol DNA polymerase 2 - 3 fold above base levels by 72 h in culture, while enzyme activity was unchanged in those cells switched to control medium alone or grown in the continued presence of nafoxidine. DNA polymerase a activity is known to increase dramatically during the S phase of the cell cycle, and inhibitors of protein or RNA synthesis have been shown to prevent this increase [68]. Thus, increased activity is believed to result from new enzyme synthesis, and our demonstration that estrogen can stimulate DNA polymerase suggests that estrogens can induce MCF-7 cells to initiate DNA replication.

We have further examined the mechanism of estrogen action in MCF-7 cells by studying estradiol effects on the dynamics of estrogen receptor regulation [69]. Within minutes after addition to the cells in vivo, estradiol enters the cell and binds to unoccuplied receptors, resulting in a rapid depletion of the unfilled receptors in the cytoplasm and nucleus. The newly formed hormone-receptor complex is translocated to sites in the nucleus from which it can only be extracted with high salt buffers. Beginning 30 rain after addition of estrogen, and continuing for 3 - 5 h, a progressive depletion of approximately 70% of the nuclear hormone-receptor complex occurs without reappearence of new receptor. We have termed this depletion of nuclear receptor 'receptor processing'. Thereafter, estrogen- bound nuclear receptors stabilize at a new state level as long as the cells are exposed to estrogen. This is illustrated in Fig. 2. We also find that the extent of translocation of unfilled receptors to the nucleus and the amount of receptor processing are dependent on the dose of estrogen [69]. With increasing doses, cytoplasmic receptor sites are proges- sively depleted which parallels an increase in filled nuclear receptors. As more receptor sites are filled and translocated to the nucleus, the 'processed' or steady state level of nuclear receptors progressively falls (i.e. total receptor loss is greater with increasing doses of estrogen). There is a limit to the amount of receptor lost in this fashion. At estrogen doses above 1 nM, increasing the dose does not promote further loss of receptors, the receptor loss remains the same (70%). Even if the hormone is removed, we find that estrogen retention to the nuclear receptor sites is prolonged, to the extent that some estradiol remains bound to nuclear sites 8 days after removal of estrogen from the medium. Never- theless, removal of estrogen leads to reappearance of unfilled cytoplasmic receptor sites. Restoration of unfilled estrogen receptor proteins cannot therefore be explained by a redistribution of existing receptor molecules in the nucleus but must rather represent de novo synthesis of receptor.

470

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Receptor processing is not due to sequestration of molecules to nuclear sites inaccessible to salt extraction nor is it due to redistribution of receptors to cytoplasmic compart- ments, but represents a true loss of total cellular estrogen binding sites. After incubating cells for 5 h with estradiol we were unable to detect any additional specific binding sites by a direct estradiol exchange assay with whole nuclei. Furthermore, when cells were incubated for 5 h with radiolabeled estradiol, specific binding was not detected in the cytosol or microsomal fractions.

Progesterone receptor, as previously demonstrated (Fig. 1), is regulated by estradiol in MCF-7 cells. When progesterone receptor induction is examined as a function of estradiol concentrat ion, we find that stimulation of progesterone receptor closely parallels the extent of binding to unfilled estrogen receptor sites and accumulation of the estrogen- receptor complex in the nucleus. It was also observed that the extent of estrogen receptor processing closely parallels progesterone receptor induction [16]. Fig. 3 illustrates this relationship by comparing the amount of estrogen receptor processing at different estradiol concentrations, with the amount of progesterone receptor induced. Stimulation of progesterone receptor is incomplete at low estradiol doses, when receptor binding and processing are also incomplete. Progesterone receptor induction reaches a maximum at 0.1 nM when receptor processing is maximal, and it is not increased further by accumulation of unprocessed bound nuclear receptor at higher doses. These relationships suggest that estrogen stimulation of progesterone receptor is mediated by the estrogen receptor.

To further illustrate that progesterone receptor induction is estrogen receptor medi-

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472

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ated, estrogen receptors and progesterone receptor levels in MCF-7 cells were followed over a 12 day incubation period of either continuous exposure to estradiol or 4 days exposure to estradiol followed by withdrawal of the hormone. The results of the experi- ment are depicted in Fig. 4. Estrogen receptor processing in cells on continuous estrogen precedes progesterone receptor induction and the stimulated progesterone receptor levels are maintained as long as estradiol is present. Withdrawal of estrogen disrupts the steady state 'processed' level of nuclear receptors, which results in a rapid replenishment of total cellular estrogen receptor and simultaneous fall of progesterone receptor to basal levels. These studies suggest that estrogen receptor processing may be an essential molecular event following accumulation of nuclear receptors in the nucleus and preceding synthesis of specific estrogen regulated proteins, such as progesterone receptor.

The nature of estrogen receptor processing is not clear. To further address this problem the effects of various transciption and translation inhibitors on processing were examined [70]. It was found that processing of nuclear receptors was completely prevented by 2/JM actinomycin D added at the same time as estradiol as shown in Fig. 5. Neither the initial binding of estradiol to unfilled receptor sites nor the translocation of the hormone- receptor complex to the nucleus is affected by actinomycin. Actinomycin may be acting to block processing either directly by preventing estrogen receptor access to specific DNA sites, or indirectly by inhibiting RNA and protein synthesis. However, we have found that when act inomycin is added anywhere from 5 min to 2 h after estradiol t reatment, the cessation of estrogen receptor processing occurs within minutes so that an intermediate

473

effect of actinomycin by way of reduced protein synthesis is not likely. Furthermore, of the many inhibitors of transcription or translation tested, including cycloheximide (at 1 -2 /aM), only chromomycin A3 and actinomycin D were able to prevent nuclear processing of the receptor-estrogen complex. Both compounds behave identically as intercalators of DNA, specific for G-C base pairs. The failure of other intercalators and translation inhibitors to prevent receptor processing, again suggests that actinomycin effects do not result from inhibition of RNA and protein synthesis, but rather that actinomycin may directly block binding of the estrogen receptor to specific DNA binding sites. Since the estrogen receptor is translocated and binds to the nucleus even in the presence of actinomycin suggests that MCF-7 nuclei may contain two binding sites for the receptor- estrogen complex. We have hypothesized therefore, based on studies with actinomycin, that separate sites are involved in the initial binding of nuclear receptors to nuclei and subsequent receptor processing. Since actinomycin blocks receptor processing, the second site at which processing occurs may be a specific base region on DNA. The existence of two receptor binding sites in nuclei, one on chromatin and another on DNA, has been postulated by Schrader et al. [71] in the chick oviduct progesterone receptor system. Palmiter et al. [72] have also proposed a two step translocation mechanism involving movement of the receptor from initial non-productive chromatin binding sites to productive sites.

VI. Antiestrogens

VIA. Experimental animal models Antiestrogens are estrogen analogues which interfere with the biological effectiveness

of estrogens in target tissues. The mechanism of action of antiestrogens has been studied principally in the rat uterus, where they have been observed to inhibit estrogen induction of growth. In vitro, antiestrogens reversibly inhibit estrogen binding to the estrogen receptor [73]. Antiestrogens in vivo, however, do not function as estrogen antagonists simply by competing for estrogen receptor binding sites. Estradiol exchange assays have shown that antiestrogens in the rat uterus do bind and translocate estrogen receptors to the nucleus [74-76]. Blockage of receptor translocation is not, therefore, involved in the mechanism of antiestrogen action. Curiously, antiestrogens in the absence of estrogen may mimic estrogen action by inducing early uterotropic responses, among which are induced protein synthesis [75], stimulation of RNA polymerase activity [77], glucose metabolism [78], and even some later effects, such as protein, DNA synthesis and uterine dry weight increase [77,79,80]. Complete uterotropic responses, however, do not develop following treatment with antiestrogens and the uterus remains refractory to further stimulation by estrogens.

Although antiestrogens bind and translocate estrogen receptors, many experiments have shown apparent failure to replenish unfilled cytoplasmic receptors. By contrast, cytoplasmic receptors are replenished by 24h after estradiol treatment. The primary mechanism of antiestrogen action has thus been proposed to be the failure to stimulate replenishment of cytoplasmic estrogen receptor, which renders the uterus insensitive to subsequent estrogen action [75,79]. Antiestrogens therefore have been hypothesized to function as both agonists and partial antagonists of estrogen action [79]. The early ago- nistic properties of antiestrogens would then be a consequence of the initial translocation and retention of receptor-antiestrogen complexes in the nucleus.

There is some controversy as to whether antiestrogens do, in fact, block cytoplasmic

474

receptor resynthesis. It has been suggested that the apparent failure to replenish cytoplasmic estrogen receptor is due only to pharmacokinetic differences between antiestrogens and estrogens and not to fundamental differences in molecular mechanisms. Estra- diol is cleared very rapidly from circulation t1/2 = 30 rain [81,82], while the biological half lives of antiestrogens are several days [83]. There is evidence to show that replenishment may actually occur with antiestrogens but newly synthesized receptors do not accumulate in the cytoplasm because the high circulating levels of ligand cause continual translocation to the nucleus [81]. In opposition to this theory, Ferguson and Katzenel- lenbogen [80] have noted that a long acting estrogen which has a duration of action similar to that of most antiestrogens was, in fact, able to stimulate cytoplasmic estrogen receptor replenishment and maintain prolonged tissue growth in spite of its prolonged retention in the circulation. Probably these differences are a function of dose rather than of estrogenicity, because we found that 48 h after injection of a low dose of the antiestrogen tamoxifen, cytoplasmic estrogen receptor was fully restored to control levels and was capable of being translocated by estradiol to the nucleus [84]. We were unable to demonstrate a relationship between tamoxifen's inability to maintain stimulation of uterine growth and prolonged depletion of cytoplasmic estrogen receptor. Failure to stimulate estrogen receptor replenishment thus appears not to be a necessary feature of antiestrogen action.

Clark et al. [74] first reported that the receptor-nafoxidine complex is apparently retained in the nucleus for a prolonged period of time compared to the receptor-estradiol complex, which is rapidly removed or 'processed' as discussed earlier. This, and similar observations including our own, led to the proposal that continued 'processing' of the nuclear receptor may be an active step required for continual estrogen action, and that antiestrogen action might be explained by a defect in processing of its receptor complex. However, current results (Chamness and Bromley, unpublished data) suggest that in the rat uterus processing of receptor-antiestrogen complexes is normal, with prolonged eleva- tion of nuclear receptor levels being due to continued replenishment and translocation as suggested above.

It is readily apparent that much of the experimental data on antiestrogen action in the rat uterus is contradictory, and the precise mechanism by which antiestrogens antagonize the action of estradiol remains unclear. However, based on the current literature, antiestrogens would appear to antagonize estrogen action by producing a receptor complex which is unable to bind or properly interact with every segment of the genome specific for the receptor-estrogen complex. Thus, full estrogenic responses are not achieved. Little is known, however, of the differences between receptor-estrogen and receptor-antiestrogen complexes in the nucleus which might account for the differences in their activity, other than the nuclear binding of antiestrogen-receptor complexes based on salt extract- ability [85-87] may be different from the nuclear binding of the receptor-estrogen complex.

Antiestrogen action has also been studied in the DMBA-induced rat mammary tumor. Many similarities between antiestrogen action in the rat uterus and in DMBA tumors have been observed. Antiestrogens can block the in vivo uptake of [3H]estradiol [88] by DMBA tumors, and estradiol exchange assays have shown that antiestrogens compete for estrogen receptor sites as well as translocate cytoplasmic receptors to the nucleus [89, 90]. As with antiestrogenic effects in the rat uterus, tamoxifen stimulates transient estrogenic effects in DMBA tumors; following in vivo administration, both tamoxifen and estradiol stimulate an early replenishment of cytoplasmic receptor [90] and an increase in

475

RNA polymerase activity [91 ]. Tamoxifen, however, is unable to sustain either response and it appears to render the tumor insensitive to further estrogen action, since estrogen does not accumulate in the nucleus when administered subsequent to antiestrogen treatment [90].

Antiestrogen effects on the growth of rat mammary DMBA-induced tumors have been studied. The antiestrogens clomiphene, nafoxidine and tamoxifen have been shown to cause inhibition of tumor growth [92-95] , though there is a single report of growth pro- rooting activity [96[. Also, induction of new tumors was reportedly prevented by nafoxidine [97]. Like estrogens, antiestrogens have been implicated in tumor induction; Clark and McCormack [98] reported that a single injection of clomiphene or nafoxidine in new- born rats caused multiple reproductive tract abnormalities including some malignancies.

There is evidence that antitumor effects of antiestrogens in the rat may be mediated by the estrogen receptor directly at the tissue level rather than by effects on synthesis of other trophic hormones [81,99,100]. There appears to be a correlation between tile estrogen receptor content in DMBA tumors and the probability of tumor regression in response to antiestrogen. Antiestrogens may, however, also influence tumor growth indirectly by affecting either estrogen synthesis or estrogen stimulation of pituitary prolactin secretion since tamoxifen in the rat can inhibit ovarian estrogen synthesis [101,102]. However, it can only partially block estrogen stimulation of pituitary prolactin release [97,103]. Although these indirect effects may facilitate antiestrogen action, no correlation has been demonstrated between tumor response to antiestrogen and serum levels of estrogen and prolactin [81,94].

VIB. Human breast cancer cells in tissue culture

Antiestrogens in the MCF-7 cell line have been shown to compete with estradiol for specific estrogen receptor sites [64,69,104], but they bind to receptor with approximately a 20 to 100 fold lower affinity than estradiol. Direct binding studies with radiolabeled tamoxifen have shown that the number of tamoxifen and estrogen binding sites are equivalent [104]. As in the rat uterus and in DMBA mammary tumors, antiestrogens translocate the MCF-7 cytoplasmic estrogen receptor to the nucleus.

In contrast to the situation in experimental animals, in the human breast cancer cell lines MCF-7 and ZR75-l, the antiestrogens tamoxifen, nafoxidine, CI-628 and clomiphene strongly inhibit both macromolecular synthesis and growth in the absence of estrogen [104]. No concentration of any antiestrogen tested was observed to stimulate either growth or macromolecular synthesis in these cell lines. Antiestrogen effects on MCF-7 cells are apparently mediated by the estrogen receptor system and are not due to nonspecific or toxic effects to the cell, since antiestrogens are without effect on cells which lack an estrogen receptor, and since the degree of antiestrogen binding to receptor parallels the degree of inhibition of macromolecular synthesis. Also, simultaneous incubation of estradiol and antiestrogens prevents the inhibitory effects of antiestrogens, and once established, antiestrogen effects are completely reversed by the subsequent addition of estradiol.

Although antiestrogen effects in the rat uterus and MCF-7 cell line appear to be mediated by the estrogen receptor, the response to antiestrogens in the MCF-7 cell is some- what different from that observed in the rat uterus. Antiestrogens appear to be strictly inhibitory in MCF-7 cells, even in the absence of any estrogen, while they stimulate weak estrogenic responses in the rat uterus or DMBA tumors and antagonize subsequent stimulation by active estrogens. Whether these dissimilar effects represent fundamentally dif-

476

ferent mechanisms of antiestrogen action in normal target tissue compared to human breast cancer is not certain. Differences could be contributed simply by the selection pro- cesses during adaptation of the cells to tissue culture conditions. These dissimilarities could also reflect differences between proliferative and non-proliferative tissues, since in the absence of circulating estrogens, the uterus in vivo is in a non-proliferative state such that it may not be possible to further inhibit cells with antiestrogens. MCF-7 cells, however, are not estrogen-dependent; they are rather estrogen-responsive and continue prolif- erating in the apparent absence of any estrogens. Because cell growth and macromolecular synthesis are suppressed by antiestrogens well below the levels of cells grown in control medium alone without estrogens, Lippman et al. [104] have hypothesized that MCF-7 cells in the absence of estrogen may synthesize basal levels of regulatory proteins which are required for continued growth and viability of the cells. Estrogens could then stimulate growth by increasing the rate of synthesis of these regulatory proteins, whereas antiestrogens might block their synthesis resulting in inhibition of cell growth. Alternatively, MCF-7 cells grown on hormone free medium may retain very small levels of endogenous estrogen, which might provide a basal growth stimulus. Addition of antiestrogen would then chase endogenous estrogen from nuclear receptor sites, resulting in cell growth inhibition.

The antiestrogens nafoxidine and tamoxifen were studied for their effects on estrogen receptor translocation and processing in MCF-7 cells [69]. Fig. 6 shows these effects. Both estrogens and antiestrogens initially bind and deplete unfilled estrogen receptor sites, which accumulate as bound receptor in the nucleus. It is the subsequent processing of receptor in the nucleus that differs. Cells incubated with tamoxifen process receptor to a lesser degree than those incubated with estradiol, while cells incubated with nafoxidine demonstrate no receptor loss at all.

We have proposed that estrogen receptor processing in the nucleus may be an essential step for estrogen action. Therefore, the fact the receptor fails to process when bound with nafoxidine may be a significant finding. This difference in nuclear mechanisms for estrogens and antiestrogens may account for their differing biological activities. If so, the differences in processing between nafoxidine and tamoxifen should be reflected in differing estrogenicities.

We have indeed observed that tamoxifen mimics at least one estrogen effect, the induction of progesterone receptor, while nafoxidine has little or no effect on progesterone receptor. Fig. 7 shows the effects of several concentrations of these antiestrogens on estrogen receptor processing and induction of progesterone receptor, an estrogenic response, in MCF-7 cells. We see that receptor complexed to nafoxidine does not process and likewise has no effect on progesterone receptor, whereas the nuclear receptor is processed to a small extent when bound to tamoxifen and considerably increases progesterone receptor until doses are reached which kill the cell. (This clearly modifies the previ- ous findings that antiestrogens have no estrogenic activity in MCF-7 cells in the absence of estradiol.) Our data with antiestrogens thus further supports the contention that processing of the receptor from the nucleus may be an active step in the mechanism of estrogen receptor action.

In the rat uterus, models of estrogen action have been proposed having at least two nuclear binding sites for the estrogen-receptor complex, only one of which is accessible to the antiestrogen-receptor complex [86,105,106]. We have similarly proposed a two site model for estrogen action in MCF-7 cell nuclei, suggesting that processing of the receptor- estrogen complex occurs at a second binding site and is required for eliciting estrogenic responses. The dual actions of tamoxifen seem compatible with such a two site model.

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VIC. Clinical trials Antiestrogens have been used in clinical trials in the treatment of breast cancer. Since

circulating estrogens may be an important factor in stimulating breast tumor growth in humans, the rationale for using antiestrogens has been to block estrogen action at the level of the cancer tissue itself, thereby accomplishing the same end as endocrine ablative therapy, yet eliminating the trauma of surgery. The antiestrogens which have been used in clinical trials are clomiphene, tamoxifen and nafoxidine. The cumulative patient tumor response to antiestrogen treatment from a number of studies [107-119] is presented in Table VI. Antiestrogens have been found to be effective, and the response rate is similar to that of other endocrine manipulations.

In some breast tumor biopsies, antiestrogens have been shown to inhibit estrogen binding to its receptor, both in vivo [120] and in vitro [121,122]. Several case studies have reported a correlation between objective tumor remission to antiestrogen treatment and the presence of estrogen receptor in the tumor biopsy [ 116-117,123,124]. Although the preponderance of data suggests there is such a correlation, the correlation is not suffi- ciently strong to prove that the primary antitumor action of antiestrogens is necessarily mediated by the tumor estrogen receptor. It is possible that antiestrogens act at other sites to effect tumor regression, although patients whose tumors have responded to tamoxifen are reported to have unchanged plasma levels of estradiol and prolactin [ 114] and menstrual cycles in premenopausal women are unchanged [124]. Possible indirect effects of antiestrogens in promoting mammary tumor regression may be indicated by both clinical studies and experimental animal studies. Tamoxifen partially inhibits estrogen stimulated prolactin secretion in rats [103], acts directly on the ovary in pregnant rabbits to inhibit the leuteinizing action of estradiol [125], and can block the stimulatory effect of gonadotropins on estrogen synthesis in the rat ovary [126]. In humans, however, tamoxifen appears to have little effect on circulating estrogen levels and has been shown to have only minimal effects on secretion of pituitary prolactin and gonadotropins [127].

Most patients receiving antiestrogen therapy have been postmenopausal. The response rate as shown in Table VI in a small series of premenopausal patients was low, which emphasizes that the endocrine status of a patient may influence the action of antiestrogens in promoting tumor regression.

Despite the fact that antiestrogens may influence peripheral endocrine systems and may have carcinogenic potential, especially in fetal life [98], they produce a tumor response rate equivalent to that of more traditional hormonal therapies and should be of considerable value for the treatment of breast cancer. In some instances, antiestrogen treatment may, in fact, be more efficacious than other forms of endocrine therapy. There is ample evidence to show that estrogens can be produced ectopically [35,128,129] by the breast tumor itself, in which case ovariectomy or adrenalectomy would not effec- tively eliminate body production of estrogens, while antiestrogens might be able to block the effects of these estrogens on tumor growth. For a more detailed discussion of antiestrogen action in breast cancer the reader should refer to the recent review by Horwitz and McGuire [130].

VII. Current concepts and future developments

Although the role of cytoplasmic estrogen receptors in the prediction of endocrine responsiveness in breast tumors is well established, the simple presence or absence of

480

estrogen receptors does not absolutely indicate whether growth of a particular breast tumor is sensitive to estrogens. More extensive and more mechanistic studies are needed to fully understand why tumors containing estrogen receptor do not respond consistently to endocrine manipulations. This is an important consideration and has been a major goal of the studies in our own and numerous other laboratories.

One approach is suggested by the finding that the probability of tumor regression correlates better with a quantitative than with a qualitative assessment of estrogen receptors. This lends support to the concept that malignant transformation results in a heterogenous population of estrogen receptor-containing cells. It has not been possible by conventional biochemical assays to establish whether a tumor with high estrogen receptor values contains more cells within a mixed population containing estrogen receptors or whether a particular cell type has a higher concentration of receptor sites. This is an important ques- tion, since the biological behavior of tumor cells following endocrine therapy might depend on whether a tumor contained a mixed population of cells with respect to estrogen receptors. If this were the case, one would expect cells containing estrogen receptors to regress following endocrine treatment, leaving only autonomous, estrogen receptor- negative cells. These cells would then continue to grow leading to eventual re-appearance of the tumor. The relapsed tumor should be relatively hormone resistent and be more likely to respond to chemotherapy. Clinical trials indicate, however, that this is not always the case. The concept, therefore, that a breast tumor contains two populations of cells wtfich either do or do not regress following endocrine therapy is overly simplistic. To answer some of these questions regarding the population of neoplastic epithelial cells containing estrogen receptors, cytochemical methods are now available for detecting estrogen receptors in human breast tumor biopsies [131-135]. These methods have the distinct advantage of permitting simultaneous, histological examination of intact tissues and identification of estrogen receptors in individual cells. Cytochemical assays have shown, in fact, that the majority of breast tumors do contain heterogeneous cell populations of estrogen receptor-positive and -negative cells, mixed in various proportions. The intensity of cytochemical staining for estrogen receptor in individual cells also appears to be variable. Tumors displaying homogeneous cell types (all cells exhibiting no receptor binding, or all cells exhibiting binding) are reportedly rare. It has been proposed from the results of cytochemical studies that all breast tumors may contain some estrogen receptor- positive cells and it may be more appropriate, therefore, to classify tumors by their proportion of estrogen receptor-negative and -positive cells rather than by an absolute estrogen receptor-negative or -positive evaluation [133]. An important application for cytochemical estrogen receptor assays as suggested by Lee [133] is to examine the proportion of estrogen receptor-containing cells in a breast tumor biopsy before and after endocrine therapy in order to answer fundamental questions as to the biological behavior of hormone dependent tumors.

Taking another approach, we have proposed that an ideal biochemical marker of a tumor's sensitivity to hormones would be a specific end product of hormone action. The presence of progesterone receptor, therefore, which has been shown to be under estrogen control, would indicate that the transformed cells continued to synthesize a specific protein under hormonal control and that tumor growth presumably would also be sensitive to estrogens. Although the response rate to endocrine therapy in tumors containing both estrogen receptor and progesterone receptor is indeed much greater than in any other group, we do not know whether growth and progesterone receptor synthesis are actually linked by a common estrogen sensitive pathway. There is evidence in experimental animal

481

models to show that in some instances estrogen control of growth and progesterone receptor may not be linked. We observed growth of 2 rat DMBA-induced tumors to be autonomous, yet progesterone receptor was estrogen dependent [48]. More recently, Margot et al. [136] observed that progesterone receptor was regulated by estrogen in the estrogen-independent MTW-9B transplantable mammary tumor. To approach the ques- tion of whether estrogen control of tumor growth and other specialized functions such as progesterone receptor synthesis are intimately associated, it may be necessary to identify other estrogen regulated proteins in breast tumor ceils. For instance, breast tumor cells may exhibit a specific estrogen inducible protein or group of proteins analogous to the induced protein of the rat uterus [137,138]. In the rat uterus, induced protein appears very early in the response sequence and has been suggested to play a role in the further action of the hormone. Demonstration of specific estrogen inducible proteins in breast tumors may be useful not only in developing new predictive tests for hormone dependence of a tumor but also to our understanding of the mechanisms involved in hormone control of breast carcinomas.

Since the sequence of biosynthetic events in the action of estrogens is complex, it is possible that a defect in the biochemical pathways at any point distal to estrogen binding with the receptor could account for the insensitivity of estrogen receptor-positive tumors which fail to respond to endocrine therapy. There is precedent for this assumption. A mouse lymphoma cell line has been cloned which contains cytosol glucocorticoid receptors, but is unresponsive to glucocorticoids [139]. Insensitivity to glucocorticoids apparently arises as a result of a defect in receptor binding to nuclei and not to receptor activity for glucocorticoid binding. The defect may reside in the receptor protein itself, pos- sibly as a result of mutation in the genetic code for the receptor, since glucocorticoid receptors isolated from the wild-type, sensitive cells are able to bind to nuclei isolated from the resistent cells yet the receptors from the resistent cell line do not bind to nuclei from sensitive ceils [140]. It seems entirely possible that a similar defect in the estrogen receptor system may occur in some breast tumors. Identification of such biochemical lesions distal to estrogen binding with receptor would be an invaluable aid to predicting the hormone responsiveness of a particular breast tumor. One could envision developing a routine in vitro assay, in addition to estrogen receptor measurements, for specific biochemical markers in the molecular pathways mediating estrogen action. As yet, no one has clearly demonstrated such a specific defect in breast tumors. Spelsberg et al. [141] proposed that transformed cells which contain steroid receptors but do not respond to steroids may have masked or defective nuclear 'acceptor' sites for binding the translocated steroid-receptor complex. One preliminary study [142] has suggested that hormone dependent human breast tumors do contain acceptor sites for the estrogen receptor, while no acceptor sites were detected in chromatin of autonomous tumors. It could be of value, therefore, to develop 'acceptor' site assays in order to examine the correlation between the presence or absence of acceptor sites with the presence of estrogen receptor and the tumor response to endocrine manipulation.

Another study related to the problem of defective estrogen receptor systems was that of Nenci et al. [135] who by immunofluorescent detection of estrogen receptors, demonstrated that the nuclear translocation of receptors may be defective in some human breast tumors. Tumor sections incubated at 4°C resulted in most cells exhibiting fluorescence only in the cytoplasm. At elevated temperatures, some cells showed an increase in nuclear staining while others, retained cytoplasmic staining but failed to transfer the fluorescent antibody to the nucleus. It was also noted that certain breast tumor cells displayed a peri-

482

nuclear concentration of the fluorescent antibodies similar to a pattern observed in the uterus of very immature rats [135], suggesting that during ontogenic and early postnatal development, analogous changes in permeability of the nuclear membrane to estrogen receptor may occur to give protection to the tissue against circulating estrogens.

So far, the discussion of predicting breast tumor hormone dependence has focused on estrogen receptor and other indirect correlates. One could also consider more direct mea- sures, such as explanting tumor cells into primary tissue culture and measuring estrogen effects on growth parameters, such as DNA synthesis, thymidine incorporation, etc. This could provide valuable information in addition to estrogen receptor and progesterone receptor measurements. This would not be a feasible system for obtaining routine clinical data, but could be of vlaue as an experimental model. Alternatively, tumors could be transplanted to experimental animals such as athymic 'nude' mice and the response to endocrine manipulations determined in vivo. This technique, however, has been shown with two mammary adenocarcinomas to run the risk of changes in biological response characteristics and histological appearance after repeated transplantations [143]. No changes occurred, however, in the early passages [143] and changes occurring during serial transplantations may be peculiar to each tumor, since there are reports of some tumors which show no change in cytological characteristics or in chromosome number over a period of 27 to 56 passages [144]. A human mammary tumor xenograft-nude athymic mouse may be a viable system to test the short term effects of endocrine manipulations on tumor biology.

The emphasis on estrogen action and estrogen receptors should not cause one to over- look the fact that in mammary tissue, the biological role of one hormone cannot be completely understood apart from the actions and interrelationships of other hormones. Since an estrogen receptor is only one part of the complex hormone system regulating mammary tissue growth and function, the simultaneous measurement of other hormone receptors may be helpful in predicting tumor response to hormone manipulation. This is particularly important because current studies clearly show that certain hormones are capable of regulating receptor synthesis and/or functions of other hormones. This may begin to explain the complex hormonal requirements of normal and neo-plastic breast tissues and why it is has been difficult to consistently predict the biological behavior of a particular tumor based on biochemical markers for a single hormone. The MCF-7 cell line, which contains four classes of steroid receptor, may be an ideal model for studying the interre- lationship of the steroid hormones which influence breast cancer growth.

Another hormone demonstrated to play an important role in regulation of mammary tumor growth in both humans and experimental animal models is progesterone. In the DMBA-injected rat, progesterone has been implicated as the hormone responsible for pregnancy-promoted tumor growth [145-150], while from clinical studies, progesterone has been proposed to have an inhibitory effect on breast tumor growth [151-153]. Pro- gesterone receptor measurements, therefore, may be potentially useful as a marker of a tumor's sensitivity to the actions of progesterone, as well as being an end product of estrogen action. By the same reasoning leading to progesterone receptor measurement as an end product of estrogen action, estradiol dehydrogenase, which is regulated by progesterone in the rat uterus [ 154], may be a good biochemical marker for progesterone action in breast tumors.

Considerable progress has now been made in understanding the mechanisms of endocrine regulation of breast tumor growth. This has been contingent upon the demonstration of specific receptor proteins for each hormone. Further investigations into steroid

483

receptor mediated functions should lead to a more complete understanding of the mech-

anisms underlying hormone control of breast cancer and to new approaches in the endo-

crine management of breast cancer patients.

Acknowledgements

We would like to thank Dr. Kathryn B, Horwitz and Dr. Roberto E. Garola for provid-

hag data used in portions of this review. Data from the author's laboratory was supported

ha part by The National Institutes of Health (CA11378, CB23862, CA09042) and The

American Cancer Society.

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estrogen and progesterone receptor proteins in breast cancer

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